Steroid receptors are dependent on multiple interactions with the molecular chaperone machinery to maintain receptor in a functionally quiescent state that is competent for hormone binding and subsequent activation as a transcription factor. Functionally mature receptor complexes consist of a receptor monomer, an Hsp90 dimer, the cochaperone p23, and one of the immunophilin class of Hsp90-binding cochaperones. Over the last few years, evidence has accumulated that two of the immunophilins, FKBP52 and FKBP51, can increase or decrease, respectively, the hormone binding affinity (HBA) of glucocorticoid receptor (GR). The main hypothesis to be tested in this proposal is that FKBP51 and FKBP52 function in an opposing fashion to modulate cellular responsiveness to glucocorticoids. In testing this hypothesis, we intend to gain a mechanistic understanding of how each FKBP functions and what alterations occur in GR that result in HBA changes. Preliminary evidence points to FKBP peptidylprolyl isomerase activity as an important factor in alterations of GR HBA. We will pursue the following Specific Aims: Aim 1 - Demonstrate that FKBP51 in opposition to FKBP52 can lower GR HBA (hormone binding affinity) in ceils. We will use a genetically robust yeast model for chaperone function in GR signaling to gain a cellular perspective on FKBP actions. Findings in the yeast model will inform additional experiments in human cells. Aim 2 -Identify the structural and mechanistic differences in FKBP51 and FKBP52 that influence GR HBA. FKBP51 and FKBP52 are structurally similar, multi-domain proteins that impact GR function in opposite ways. Crystallographic data and in vivo functional assays will be used with mutational approaches for detailed structure/function mapping of each FKBP. Aim 3- Identify structural/ changes in GR that correlate with increase/decrease in HBA. We propose that a proline-containing sequence bordering the GR hormone binding pocket is subject to structural alterations mediated by receptor-associated FKBP. A combination of in vitro and in rive approaches will be used to test this hypothesis. Achieving the stated Specific Aims will provide a unique insight on the roles FKBP immunophilins play in regulating cellular responses to glucocorticoids. Identifying mechanisms by which cells can be sensitized or desensitized to glucocorticoids has direct implications for understanding and perhaps therapeutically altering glucocorticoid-regulated pathways that are important in homeostasis and disease.